This invention relates to the general fields of passive mucosal immune protection, and of poly-Ig immune reagents and techniques. We use the term poly-Ig to refer to the polymeric classes of antibodiesxe2x80x94i.e. IgA and IgM. IgM antibodies are generally produced at an early stage of the immune response and are not an important factor in protective mucosal immunity. Thus, the invention generally refers to polymeric Ig antibodies, and the usual and preferred antibodies for all aspects of the invention are IgA class antibodies, which normally are secreted in dimeric form and, to a lesser degree, as higher IgA polymers.
Many pathogenic bacteria and viruses initially gain entry into the body by crossing the cellular linings (epithelia) of the gastrointestinal, respiratory, or genital tracts. A specialized class of antibodies, IgA antibodies, protects these surfaces. IgA antibodies are dimeric or polymeric molecules produced by cells located in the tissues under the epithelial surfaces. They are transported by epithelial cells into mucosal secretions, where they cross-link or coat pathogens that have not yet entered the body, preventing the pathogens from contacting and adhering to epithelial cells. Thus, IgA antibodies operate on pathogens that are outside the body, and they protect by preventing entry into the body across epithelial surfaces.
The naturally occurring IgA response is triggered by antigen delivery to mucosal surfaces. The antigen enters the body through specific sampling sites (termed microfold or M cells) that effect transepithelial antigen transport to areas of the mucosal lining containing specialized, organized collections of the cells of the mucosal immune system. More specifically, as shown in FIG. 1, antigens A (shown as filled dots) in lumen 1 bind the luminal surface of M cells at site 2. The antigens are internalized and transcytosed at 3 to be released in the intra-epithelial pocket 4 containing lymphoid cells L (B and T cells) and antigen-processing/presenting cells such as macrophage cells (M).
IgA antibodies in a naturally immunized host are transported into secretions by binding to a specific receptor (called the poly-Ig receptor) on the basal (interior) surfaces of epithelial and glandular cells throughout the respiratory and digestive systems, the genital tract, and the mammary glands. See Solari and Kraehenbuhl, xe2x80x9cReceptor-Mediated Transepithelial Transport of Polymeric Immunoglobulinsxe2x80x9d, pp.269-298 in The Mammary Gland, Nelville and Daniel Eds., Plenum Publishing, Cambridge (1987); Mestecky (1987) J. Clin. Immunol. 7:265-276. Receptor-IgA complexes are transported across these cells and exocytosed onto luminal (exterior) cell surfaces where the receptor is enzymatically cleaved, releasing IgA into secretions along with a receptor fragment called secretory component (SC). See Mostov et al. (1980) Proc. Nat""l Acad. Sci. U.S.A. 77:7257-7261; Solari, R. and Kraehenbuhl, J. P. Cell 36:61-71 (1984); Kuhn and Kraehenbuhl, J. Biol. Chem. 256:12490-12495(1981). It is reported that secretory component reduces proteolytic degradation of IgA. Lindh, J., J. Immunol. 114:284-286 (1975); Brown, Neucomb, Ishizaka, J. Clin. Invest. 49:1374 (1974).
In general, existing immunization strategies which involve injection of antigens evoke production of the IgG class of antibodies that circulate systemically and neutralize pathogens after they have entered the body. Injection of antigens does not generally evoke a substantial IgA response.
Efforts to take advantage of IgA protection at mucosal barriers involve oral immunization, either for active protection of the immunized mammal or for passive protection of another mammal using mucosal secretion of the immunized mammal. Glass et al., New Eng. J. Med., 308:1389-1392 (1983); Fubara et al., J. Immunol., 111(2):395-403 (1973). Monoclonal IgA antibodies have been produced and applied directly to respiratory mucosal surfaces in an effort to protect against pathogen entry. Mazanec et al. J. Virol., 61:2624-2625 (1987).
Active immunization may involve challenge at the mucosal surface with intact (killed) bacteria or viruses. To avoid dangers that may be associated with this approach for certain pathogens, component antigens, such as immunogenic surface components of the pathogen, are applied at a mucosal surface. In some cases, the antigens have been conjugated to larger molecules. For example, the cholera toxin B subunit has been conjugated to antigens. See, Czerkinsky et al. who report oral administration of a streptococcal antigen coupled to cholera toxin B subunit in Infection and Immunity 57:1072-1077 (1989). Biodegradable microscheres have also been used as an antigen carrier. For example, Eldridge et al. Curr. Top. Microbial Immunol. 146:59 et seq. (1989) report incorporation of antigen into biodegradable microspheres. The dry protein antigen is dispersed in a copolymer matrix without chemical conjugation.
We have discovered that hydroxylated calcium phosphate (HCP) particulate is a particularly useful carrier for antigens to be applied to mucosal surfaces. The antigen-HCP conjugate is transported across epithelium where it raises a poly Ig immune response.
One aspect of the invention generally features a method for generating antigen-sensitized Ig-A-producing lymphoblasts in a mammal. In that method, an immunogen comprising an antigen or antigen mixture in association with hydroxylated calcium phosphate (HCP) particulate is administered to a mucosal surface of the mammal. In preferred embodiments of this first aspect of the invention, the antigen-sensitized lymphoblasts are recovered and immortalized to yield an Ig-A producing hybridoma.
A second aspect of the invention features a method for vaccinating a mammal (especially a human) comprising administering the above-described immunogen to a mucosal surface of the mammal.
A third aspect of the invention features an immunogen comprising an antigen or antigen mixture in association with hydroxylated calcium phosphate particles of a size suitable for transport across epithelium.
In preferred embodiments of any of the three aspects of the invention the hydroxylated calcium phosphate is in the form of microparticles suitable for transport across the epithelium. Also preferably, the antigen comprises an externally available determinant of a pathogen or of spermatozoa, such as a viral coat or envelope protein, a lipopolysaccharide or a cell-surface protein. One form of HCP is hydroxyapatite (HA), a commercially available crystalline hydroxylated calcium phosphate discussed below.
The preferred modes of administrations of the immunogen according to the first two aspects of the invention are orally, vaginally, nasally, rectally, ocularly or to the middle ear. Oral administration can provide delivery to other G.I. mucosa including the intestinal mucosa.
The invention provides an efficient, polyvalent immunogen that can adhere to the mucosa and can be transported efficiently across the epithelial barrier for presentation to the mucosal immune system. Adsorption of proteins to HCP is relatively simple, rapid and cheap, making the invention economically feasible. Moreover, HCP has a high general affinity for the antigens of interest, including proteins and other antigens, making the invention broadly applicable. HCP is generally non-toxic, as evidenced by the fact that HA is an integral component of bone, and the systemic immune system routinely encounters HA during normal bone resorption, a process that occurs constantly at a microscopic level in healthy individuals. Accordingly, pure HA presumably can be safely administered without a host immune response, and administration can be repeated as a vehicle for the same or different antigens, without an anti-vehicle immune response. Moreover HCP, particularly HA, is relatively inexpensive. HA can readily be reduced to a size suitable for transepithelial transport by M cells; and such a reduced size is suitable For ingestion by macrophages and other cells of the reticuloendothelial system, so as to enhance immune response. Finally, M-cell uptake and transport of immunogens according to the invention is relatively selective.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof.